การโคลนบางส่วนของยีน Geranylgeranyl diphosphate synthase (JcGGPPS) ของสบู่ดำและการลดการแสดงออกของยีน GGPPS ในยาสูบด้วย antisense JcGGPPS
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Abstract
Jatropha curcas is one of the potential biodiesel production plant. However, one serious problem is that the plant produces phorbol esters (PEs), a cacinogen. Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the phorbol esters biosynthesis. To study the role of GGPPS gene, the partial GGPPS gene was cloned by using RT-PCR from total RNA extracted from jatropha seed cv. Korat and with the primer specific to GGPPS gene. The DNA fragment size of 592 bp was amplified. The sequence analysis indicated that the obtained cDNA had very high similarity (99 %) to GGPPS gene of J. curcas. We named this DNA fragment as JcGGPPS. The antisense JcGGPPS gene was introduced into tobacco by Agrobacterium tumefacients strain EHA-105. The four transgenic tobacco plants were produced. The analysis of GGPPS gene expression by real-time PCR showed that the lowest expression level of endogenous GGPPS gene in transformed plants was about 3 folds lower than that of the control plant. The phorbol esters contents of transformed tobacco plants were determined by high performance liquid chromatography (HPLC). It was found that the PEs content of the transformed tobacco was also 2.5 folds lower than that of the control. It was indicated that the antisense JcGGPPS gene was an effective down-regulating in the expression of endogenous GGPPS gene and reducing the PEs content of tobacco.
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References
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